Fibronectin, one of the glycoproteins of extracellular matrix (ECM), binds to membrane spanning receptors, other extracellular matrix components, such as collagen, fibrin, syndecans, etc. Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation. It is also important for other processes, such as wound bealing and embryonic development.

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Collection of Fibronectin coating protocols

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Fibronectin coating protocol from Sigma

Optimal conditions for attachment must be determined for each cell line and application.

1. Reconstitute with 0.5 mL sterile water. Do not agitate. Allow the solution to stand 30 minutes to solubilize. A small amount of undissolved material may remain. This will not affect product performance.

2. Dilute fibronectin in sterile balanced salt solution and coat the culture surface with a minimal volume.

3. Allow to air dry for at least 45 minutes at room temperature. Excess fibronectin may be removed by aspiration, but is not necessary.

 

Fibronectin coating protocol from ABIF

1. Stock fibronectin (Sigma F-0895, 1 mg/ml Fibronectin from human plasma) should be frozen (-80oC) in aliquots that can be used within about a month. After about a month in the fridge the protein degrades and cells no longer spread well.

2. Dilute 1mg/ml stock fibronectin with PBS++to 1 g/ml for MEF or 3T3 cells or 2g/ml for CHO cells. (1 l/ml PBS or 2 l/ml PBS)

3. Place 1 ml of fibronectin solution on coverslip in glass bottom dish and leave in the fridge overnight.

4. Rinse 3X with PBS(with or without calcium and magnesium)and leave in the fridge for use within two weeks

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Fibronectin coating protocol from Jacobs

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Procedure

Day 1 (~1 hr with 1 hr waiting)

1. Slide preparation

2. Soak slides in 100% EtOH for at least five minutes (usually 20 for flow is a good number).

3. Open 20 petri dishes in the hood.

4. Bring the slides and EtOH into the hood.

5. Sterilize the flat-ended forceps in the same 100% ethanol.

6. Lean slides to dry on edge of the petri dish (face up)

7. Flip the slides after laying them all down so that the other side can dry and any ethanol that pools on the lid can dry.

8. Once all the ethanol has evaporated (there should be no wet shiny places on the slides), place the slide flat on the bottom of the petri dish.

9. Place covers on the dishes.

Fibronectin Solution

1. Prepare 10 ul fibronectin per 1 mL PBS solution (1:100). You will need 1 mL of solution per slide.

   • Volume of Fibronectin solution = [# of slides] x 1 ml/slide

2. Store for up to 6 months at 4º C

3. For 20 slides, that means 200 ul fibronectin into 19.8 ml PBS.

4. Mix well.

Coating

1. Carefully pipette 1 ml of fibronectin solution on top of each glass slide. This is a delicate process, be careful and take it slow

2. Do not allow the solution to fall over the edge of the slide. If this happens, you need to collect the liquid, sterilize the slide, dry and try again. Any liquid that falls over the side will suck the rest of the liquid out and under the slide, which we do not want.

3. Make sure the solution covers the entire slide. You may use a sterile pipette tip to smooth the liquid onto any bare spots. Surface tension will allow the liquid to stay on the slide, but again, don’t push it over the sides.

4. Cover and let the slide sit undisturbed at RT in the cell culture hood for 1 hr.

5. Plate cells

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Commercially available fibronectin coated glass coverslips

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GG-12-fibronectin:  12 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine +  fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $88.65

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GG-12-1.5-fibronectin:  12 mm diameter, # 1.5 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $98.65

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GG-14-fibronectin:  14 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $93.65

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GG-15-fibronectin:  15 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $98.65

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GG-18-fibronectin:  18 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $113.65

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GG-18-1.5-fibronectin:  18 mm diameter, # 1.5 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $123.65

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GG-20-fibronectin:  20 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $120.65

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GG-22-fibronectin:  22 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $124.65

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GG-22-1.5-fibronectin:  22 mm diameter, # 1.5 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $134.65

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GG-24-fibronectin:  24 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $134.65

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GG-25-fibronectin:  25 mm diameter, # 1 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $144.65

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GG-25-1.5-fibronectin:  25 mm diameter, # 1.5 thickness, 60 pieces of CE certified crystal clear German glass coverslips with Poly-D-lysine + fibronectin coating on both sides for cell culture and confocal fluorescence microscopy, sealed 15 pieces per bag, sterilized, ready to use $174.65

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